LOCUS       sequence-244023-        6210 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Periplasmic OM bound mNeongreen mCherry tandem, ~16 % energy 
            transfer. pNM004 - pTHV037-OmpA-SA1-177-(SA-1)-LEDPPAEL-mNG-mCh.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6210)
  AUTHORS   Meiresonne NY, van der Ploeg R, Hink MA, den Blaauwen T
  TITLE     Activity-Related Conformational Changes in d,d-Carboxypeptidases 
            Revealed by In Vivo Periplasmic Forster Resonance Energy Transfer 
            Assay in Escherichia coli.
  JOURNAL   MBio. 2017 Sep 12;8(5). pii: mBio.01089-17. doi: 
            10.1128/mBio.01089-17.
  PUBMED    28900026
REFERENCE   2  (bases 1 to 6210)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "MBio."; 
            date: "2017-09-12"; volume: "8(5). pii: mBio.01089-17. doi"; pages: 
            " 10.1128/mBio.01089-17"
COMMENT     Sequence Label: OmpA-mNG-mCh
FEATURES             Location/Qualifiers
     source          1..6210
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(53..58,59..75,76..82)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    90..106
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     95..117
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     sig_peptide     133..192
                     /codon_start=1
                     /product="signal peptide from the E. coli outer membrane 
                     protein OmpA"
                     /label=OmpA signal peptide
                     /translation="KKTAIAIAVALAGFATVAQA"
     CDS             793..1500
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYE
                     ELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFED
                     GASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIS
                     TFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQ
                     KAFTDVMGMDELYK"
     CDS             1507..2217
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1652..1670)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1920..1939
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(2254..2271)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2424..2510
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2602..2629
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2648..2739
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2740..2808,2809..3600)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2958..2977)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3771..4359
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4260..4279
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4513..4530
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4545..4685
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4611..4630)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4771..4793
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        4871..4948
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             4949..6031
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(4968..4987)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 gttttttgcg ccgacatcat aacggttctg gcaaatattc tgaaatgagc tgtttacaat
       61 taatcatccg gctcgtataa tgtgtggaat tgtgagcgga taacaatttc acacaggaaa
      121 cagaccatgg caaaaaagac agctatcgcg attgcagtgg cactggctgg tttcgctacc
      181 gtagcgcagg ccgctccgaa agataacacc tggtacactg gtgctaaact gggctggtcc
      241 cagtaccatg acactggcct catcggccaa cgcttggaga tctgccagaa cgtttgctac
      301 tacctcggga cgctcaatgg tccgacccat gaaaaccaac tgggcgctgg tgcttttggt
      361 ggttaccagg ttaacccgta tgttggcttt gaaatgggtt acgactggtt aggtcgtatg
      421 ccgtacaaag gcagcgttga aaacggtgca tacaaagctc agggcgttca actgaccgct
      481 aaactgggtt acccaatcac tgacgacctg gacatctaca ctcgtctggg tggcatggta
      541 tggcgtgcag acactaaatc caacgtttat ggtaaaaacc acgacaccgg cgtttctccg
      601 gtcttcgctg gcggtgttga gtacgcgatc actcctgaaa tcgctacccg tctggaatac
      661 cagtggacca acaacatcgg tgacgcaggc cagatcggcc agcgtccgga caacggcatg
      721 ctgagcctgg gtgtttccta ccgtttcggt cagggcgaag cagctccact tgaggatccc
      781 cccgctgaat tgatggtgag caagggcgag gaggataaca tggcctctct cccagcgaca
      841 catgagttac acatctttgg ctccatcaac ggtgtggact ttgacatggt gggtcagggc
      901 accggcaatc caaatgatgg ttatgaggag ttaaacctga agtccaccaa gggtgacctc
      961 cagttctccc cctggattct ggtccctcat atcgggtatg gcttccatca gtacctgccc
     1021 taccctgacg ggatgtcgcc tttccaggcc gcgatggtag atggctccgg ataccaagtc
     1081 catcgcacaa tgcagtttga agatggtgcc tcccttactg ttaactaccg ctacacctac
     1141 gagggaagcc acatcaaagg agaggcccag gtgaagggga ctggtttccc tgctgacggt
     1201 cctgtgatga ccaactcgct gaccgctgcg gactggtgca ggtcgaagaa gacttacccc
     1261 aacgacaaaa ccatcatcag tacctttaag tggagttaca ccactggaaa tggcaagcgc
     1321 taccggagca ctgcgcggac cacctacacc tttgccaagc caatggcggc taactatctg
     1381 aagaaccagc cgatgtacgt gttccgtaag acggagctca agcactccaa gaccgagctc
     1441 aacttcaagg agtggcaaaa ggcctttacc gatgtgatgg gcatggacga gctgtacaag
     1501 gaattcatgg tgagcaaggg cgaggaggat aacatggcta tcatcaagga gttcatgcgc
     1561 ttcaaggtgc acatggaggg ctccgtgaac ggccacgagt tcgagatcga gggcgagggc
     1621 gagggccgcc cctacgaggg cacccagacc gccaagctga aggtgaccaa gggtggcccc
     1681 ctgcccttcg cctgggacat cctgtcccct cagttcatgt acggctccaa ggcctacgtg
     1741 aagcaccccg ccgacatccc cgactacttg aagctgtcct tccccgaggg cttcaagtgg
     1801 gagcgcgtga tgaacttcga ggacggcggc gtggtgaccg tgacccagga ctcctccctg
     1861 caggacggcg agttcatcta caaggtgaag ctgcgcggca ccaacttccc ctccgacggc
     1921 cccgtaatgc agaagaagac tatgggctgg gaggcctcct ccgagcggat gtaccccgag
     1981 gacggcgccc tgaagggcga gatcaagcag aggctgaagc tgaaggacgg cggccactac
     2041 gacgctgagg tcaagaccac ctacaaggcc aagaagcccg tgcagctgcc cggcgcctac
     2101 aacgtcaaca tcaagttgga catcacctcc cacaacgagg actacaccat cgtggaacag
     2161 tacgaacgcg ccgagggccg ccactccacc ggcggcatgg acgagctgta caagtaagct
     2221 tggctgtttt ggcggatgag agaagatttt cagcctgata cagattaaat cagaacgcag
     2281 aagcggtctg ataaaacaga atttgcctgg cggcagtagc gcggtggtcc cacctgaccc
     2341 catgccgaac tcagaagtga aacgccgtag cgccgatggt agtgtggggt ctccccatgc
     2401 gagagtaggg aactgccagg catcaaataa aacgaaaggc tcagtcgaaa gactgggcct
     2461 ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag taggacaaat ccgccgggag
     2521 cggatttgaa cgttgcgaag caacggcccg gagggtggcg ggcaggacgc ccgccataaa
     2581 ctgccaggca tcaaattaag cagaaggcca tcctgacgga tggccttttt gcgtttctac
     2641 aaactctttt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     2701 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     2761 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     2821 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     2881 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     2941 agcactttta aagttctgct atgtggcgcg gtattatccc gtgttgacgc cgggcaagag
     3001 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     3061 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     3121 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     3181 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     3241 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     3301 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     3361 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     3421 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     3481 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     3541 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     3601 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     3661 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     3721 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     3781 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     3841 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     3901 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct
     3961 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     4021 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     4081 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     4141 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     4201 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     4261 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     4321 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     4381 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     4441 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     4501 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt
     4561 ctccttacgc atctgtgcgg tatttcacac cgcatatggt gcactctcag tacaatctgc
     4621 tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg
     4681 ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
     4741 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
     4801 cgtcatcacc gaaacgcgcg aggcagcaga tcaattcgcg cgcgaaggcg aagcggcatg
     4861 catttacgtt gacaccatcg aatggtgcaa aacctttcgc ggtatggcat gatagcgccc
     4921 ggaagagagt caattcaggg tggtgaatgt gaaaccagta acgttatacg atgtcgcaga
     4981 gtatgccggt gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca gccacgtttc
     5041 tgcgaaaacg cgggaaaaag tggaagcggc gatggcggag ctgaattaca ttcccaaccg
     5101 cgtggcacaa caactggcgg gcaaacagtc gttgctgatt ggcgttgcca cctccagtct
     5161 ggccctgcac gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg atcaactggg
     5221 tgccagcgtg gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta aagcggcggt
     5281 gcacaatctt ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc tggatgacca
     5341 ggatgccatt gctgtggaag ctgcctgcac taatgttccg gcgttatttc ttgatgtctc
     5401 tgaccagaca cccatcaaca gtattatttt ctcccatgaa gacggtacgc gactgggcgt
     5461 ggagcatctg gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc cattaagttc
     5521 tgtctcggcg cgtctgcgtc tggctggctg gcataaatat ctcactcgca atcaaattca
     5581 gccgatagcg gaacgggaag gcgactggag tgccatgtcc ggttttcaac aaaccatgca
     5641 aatgctgaat gagggcatcg ttcccactgc gatgctggtt gccaacgatc agatggcgct
     5701 gggcgcaatg cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggata tctcggtagt
     5761 gggatacgac gataccgaag acagctcatg ttatatcccg ccgttaacca ccatcaaaca
     5821 ggattttcgc ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca
     5881 ggcggtgaag ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa ccaccctggc
     5941 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg
     6001 acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgta agttagcgcg
     6061 aattgatctg gtttgacagc ttatcatcga ctgcacggtg caccaatgct tctggcgtca
     6121 ggcagccatc ggaagctgtg gtatggctgt gcaggtcgta aatcactgca taattcgtgt
     6181 cgctcaaggc gcactcccgt tctggataat
//
